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Coelho A.,Instituto Nacional Of Saude Dr Ricardo Jorge Insa | Picanco I.,Instituto Nacional Of Saude Dr Ricardo Jorge Insa | Seuanes F.,INSA | Seixas M.T.,INSA | Faustino P.,Instituto Nacional Of Saude Dr Ricardo Jorge Insa
Blood Cells, Molecules, and Diseases | Year: 2010

Globin genes, which encode the protein subunits of hemoglobin (Hb), are organized in two different gene clusters and present a coordinated and differential pattern of expression during development. Concerning the human α-globin gene cluster (located at chromosome region 16p13.3), four upstream highly conserved elements known as multispecies conserved sequences (MCS-R1-4) or DNase I hypersensitive sites (HSs) are implicated in the long-range regulation of downstream gene expression. However, only the absence of the MCS-R2 site (HS-40) has proven to drastically downregulate the expression of those genes, and consequently, it has been regarded as the major and crucial distal regulatory element.In this study, Multiplex Ligation-dependent Probe Amplification was used to screen for deletions in the telomeric region of the short arm of chromosome 16, in an attempt to explain the α-thalassemia or the HbH disease present in a group of Portuguese patients. We report four novel and five uncommon deletions that remove the α-globin distal regulatory elements and/or the complete α-globin gene cluster. Interestingly, one of them occurred de novo and removes all HSs except HS-10, while other eliminates only the HS-40 site, the latter being replaced by the insertion of a 39 nucleotide orphan sequence.Our results demonstrate that HS-10 alone does not significantly enhance the α-globin gene expression. The absence of HS-40 in homozygosity, found in a patient with Hb H disease, strongly downregulates the expression of α-globin genes but it is not associated with a complete absence of α-globin chain production. The study of naturally occurring deletions in this region is of great interest to understand the role of each upstream regulatory element in the native human erythroid environment. © 2010 Elsevier Inc.


De Oliveira M.M.E.,University of Minho | De Oliveira M.M.E.,Institute Pesquisa Clinica Evandro Chagas | Verissimo C.,Instituto Nacional Of Saude Dr Ricardo Jorge Insa | Sabino R.,Instituto Nacional Of Saude Dr Ricardo Jorge Insa | And 4 more authors.
Diagnostic Microbiology and Infectious Disease | Year: 2014

In this study, we characterize the first autochthone case of human sporotrichosis reported in Lisbon, Portugal. Phenotypic and genotypic characterization revealed that the infection was caused by Sporothrix globosa. We conclude that sporotrichosis may be underdiagnosed particularly in Southern Europe and suggest Portugal as an emerging area for this fungal infection. © 2014 Elsevier Inc.


Manso H.,Instituto Nacional Of Saude Dr Ricardo Jorge Insa | Manso H.,Instituto Gulbenkian Of Ciencia Igc | Manso H.,Center for Biodiversity Functional and Integrative Genomics | Krug T.,Instituto Gulbenkian Of Ciencia Igc | And 12 more authors.
Atherosclerosis | Year: 2012

Objective: Animal studies have allowed important insights into the role of the nitric oxide synthase (NOS) enzymes in atherosclerosis and hypertension, as well as in stroke. In this study we tested the hypothesis that the NOS1 and NOS3 genes, respectively encoding neuronal NOS (nNOS) and endothelial NOS (eNOS), influence stroke susceptibility and outcome after a stroke event. Methods: We conducted a case-control association study in 551 ischemic stroke patients and 530 controls to assess the role of NOS1 and NOS3 variants in stroke susceptibility. The same genes were tested for association with stroke outcome in a subset of 431 patients. Results: Four NOS1 single nucleotide polymorphisms (SNPs) (rs2293050, rs2139733, rs7308402 and rs1483757) and four haplotypes were significantly associated with stroke susceptibility after adjusting for demographic, clinical and life-style risk factors, and correcting for multiple testing using the false discovery rate (FDR) method (SNPs: 0.004< uncorrected P<0.007 and 0.036


Gomes-Alves P.,Instituto Nacional Of Saude Dr Ricardo Jorge Insa | Gomes-Alves P.,New University of Lisbon | Couto F.,University of Lisbon | Pesquita C.,University of Lisbon | And 2 more authors.
Biochimica et Biophysica Acta - Proteins and Proteomics | Year: 2010

F508del-CFTR, the most common mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, disrupts intracellular trafficking leading to cystic fibrosis (CF). The trafficking defect of F508del-CFTR can be rescued by simultaneous inactivation of its four RXR motifs (4RK). Proteins involved in the F508del-CFTR trafficking defect and/or rescue are therefore potential CF therapeutic targets. We sought to identify these proteins by investigating differential proteome modulation in BHK cells over-expressing wt-CFTR, F508del-CFTR or the revertant F508del/4RK-CFTR. By 2-dimensional electrophoresis-based proteomics and western blot approaches we demonstrated that over-expression of F508del/4RK-CFTR modulates the expression of a large number of proteins, many of which are reported interactors of CFTR and/or 14-3-3 with potential roles in CFTR trafficking. GRP78/BiP, a marker of ER stress and unfolded protein response (UPR), is up-regulated in cells over-expressing either F508del-CFTR or F598del/4RK-CFTR. However, over-expression of F508del/4RK-CFTR induces the up-regulation of many other UPR-associated proteins (e.g. GRP94, PDI, GRP75/mortalin) and, interestingly, the down-regulation of proteasome components associated with CFTR degradation, such as the proteasome activator PA28 (PSME2) and COP9 signalosome (COPS5/CSN5). Moreover, the F508del-CFTR-induced proteostasis imbalance, which involves some heat shock chaperones (e.g. HSP72/Hpa2), ER-EF-hand Ca2+-binding proteins (calumenin) and the proteasome activator PA28 (PSME2), tends to be 'restored', i.e., in BHK cells over-expressing F508del/4RK-CFTR those proteins tend to have expression levels similar to the wild-type ones. These findings indicate that a particular cellular environment orchestrated by the UPR contributes to and/or is compatible with F508del/4RK-CFTR rescue. © 2010 Elsevier B.V. All rights reserved.


PubMed | University of Lisbon and Instituto Nacional Of Saude Dr Ricardo Jorge Insa
Type: Journal Article | Journal: Food chemistry | Year: 2015

The method for separation and quantitative determination of the main carotenoids in food by high-performance liquid chromatography (HPLC) was in-house validated. Tomato (Lycopersicon esculentum M.) as food matrix was used to demonstrate its linearity, repeatability, intermediate precision, detection and quantification limits, sensitivity and bias. In addition, stability of carotenoids was studied in function of temperature and time. Method accuracy was quantified through measurement uncertainties estimate based on this validation study. Furthermore, a study was conducted to evaluate variability coming from location in an experimental field composed by 12 subfields. The use of two metal free reverse phase columns and an organic mobile phase based on acetonitrile, methanol and dichloromethane enabled the separation of the six target compounds (trans--carotene, trans--carotene, -cryptoxanthin, all-lycopene, lutein, zeaxanthin) within a 30min run; detection at 450nm and external calibration allowed the quantification of the analytes. Carotenoids concentration and measurement uncertainty, in mg/100g, in tomato varieties lido and for salad were, respectively, 1.00.14 and 0.390.056 for trans--carotene, 82.0 and 2.30.57 for all-lycopene and 0.100.017 and 0.080.015 for lutein; trans--carotene, -cryptoxanthin and zeaxanthin were not detected in both varieties (detection limits, in g/100g, 0.81, 0.57 and 0.77, respectively). For -carotene and lutein, uncertainty associated with the entire process including small-scale within-region variation was statistically different, at a significance level of 5%, from measurement uncertainty (which includes sampling in the laboratory).


Schmiedt M.-L.,Finnish National Institute for Health and Welfare | Schmiedt M.-L.,Institute for Molecular Medicine Finland | Bessa C.,Instituto Nacional Of Saude Dr Ricardo Jorge Insa | Bessa C.,Abel Salazar Biomedical Sciences Institute | And 7 more authors.
Human Mutation | Year: 2010

Neuronal ceroid lipofuscinoses (NCLs) represent a group of children's inherited neurodegenerative disorders caused by mutations in at least eight different genes. Mutations in the CLN5 gene result in the Finnish variant late infantile NCL characterized by gradual loss of vision, epileptic seizures, and mental deterioration. The CLN5 gene encodes a lysosomal glycoprotein of unidentified function. In this study, we have used both transient and stable expression systems for the characterization of CLN5, focusing on the localization, processing, and intracellular trafficking. We show that CLN5 is proteolytically cleaved, and that the mature polypeptide is transported to the lysosomes. Our data provide the first evidence that soluble CLN5 protein can also undergo mannose-6-phosphate receptor-independent trafficking to the lysosomes. We studied the localization and maturation of the CLN5 carrying the previously uncharacterized vLINCL disease causing mutations in HeLa cells. All analyzed disease mutations disturb the lysosomal trafficking of the mutated CLN5 proteins. The level of lysosomal targeting does not correlate, however, to disease onset, indicating that CLN5 may also function outside lysosomes. This study furthers our understanding of the basic properties of the CLN5 protein, necessary for the characterization of the consequences of disease mutations and for the planning of future therapies for vLINCL. © 2010 Wiley-Liss, Inc.


Bettencourt A.,Abel Salazar Biomedical Sciences Institute | Silva A.M.,Abel Salazar Biomedical Sciences Institute | Silva A.M.,Centro Hospitalar Do Porto Hospital Of Santo Antonio Chp Hsa | Carvalho C.,Abel Salazar Biomedical Sciences Institute | And 5 more authors.
Journal of Neuroimmunology | Year: 2014

Killer Immunoglobulin-like Receptor (KIR) genes may influence both resistance and susceptibility to different autoimmune diseases, but their role in the pathogenesis of Multiple Sclerosis (MS) is still unclear. We investigated the influence of KIR genes on MS susceptibility in 447 MS Portuguese patients, and also whether genetic interactions between specific KIR genes and their HLA class I ligands could contribute to the pathogenesis of MS. We observed a negative association between the activating KIR2DS1 gene and MS (adjusted OR. = 0.450, p. = 0.030) independently from the presence of HLA-DRB1*15 allele. The activating KIR2DS1 receptor seems to confer protection against MS most probably through modulation of autoreactive T cells by Natural Killer cells. © 2014 Elsevier B.V.


Ribeiro D.,Instituto Nacional Of Saude Dr Ricardo Jorge Insa | Duarte A.J.,Instituto Nacional Of Saude Dr Ricardo Jorge Insa | Amaral O.,Instituto Nacional Of Saude Dr Ricardo Jorge Insa
Genetic Testing and Molecular Biomarkers | Year: 2011

Tay-Sachs disease is a rare autosomal recessive neurodegenerative disorder that results from mutations in the HEXA gene, leading to β-hexosaminidase A (HexA) α subunit deficiency. An unusual variant of Tay-Sachs disease is known as the B1 variant. Previous studies indicated that, in northern Portugal, this is not only the most common variant but also one of the most prevalent lysosomal storage diseases. Additionally, this variant might also show a higher prevalence in populations of Portuguese and Spanish ancestry. A single mutation is invariably present in at least one of the alleles of B1 variant patients, HEXA mutation c.533G > A. To implement a method for c.533G > A testing in individuals and populations, we have optimized two distinct mutation analysis techniques, one based on restriction fragment length polymorphism analysis and the other based on allelic discrimination. We present the comparison of both methods and their advantages. Mutation screening by allelic discrimination proved to be particularly useful for the studying of large samples of individuals. It is time saving and highly reproducible, and under the conditions used, its cost is lower than the cost of polymerase chain reaction-based restriction fragment length polymorphism analysis. © 2011, Mary Ann Liebert, Inc. 2011.


da Fonseca B.M.,University of Beira Interior | Moreno I.E.D.,University of Beira Interior | Magalhaes A.R.,University of Beira Interior | Barroso M.,Instituto Nacional Of Medicina Legal Delegacao Do Sul | And 5 more authors.
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2012

A new, simple and sensitive method was described for the simultaneous determination of nicotine, cotinine and trans-3'-hydroxycotinine in oral fluid samples using solid-phase extraction and gas chromatography/tandem mass spectrometry (GC-MS/MS). This technique was developed using only 0.2. mL of sample, and deuterated analogues were used as internal standards. The method was found to be linear between 0.5 and 1000. ng/mL, with determination coefficients higher than 0.996 for all analytes. Intra- and interday precision and accuracy were in conformity with the criteria normally accepted in bioanalytical method validation. All analytes were stable in the samples for at least 24. h at room temperature, for at least 72. h at 25 °C in processed samples and for at least three freeze/thaw cycles. Absolute recoveries ranged from 89 to 92% for all analytes. GC-MS/MS has demonstrated to be a powerful tool for the simultaneous quantitation of the analytes, providing adequate selectivity and sensitivity. In addition, its performance characteristics allow its routine use in the analysis of biomarkers of tobacco smoke exposure, extending the window of analyte detection in nicotine cessation programs, using a sample amount as low as 0.2. mL of human oral fluid. © 2012 Elsevier B.V.


PubMed | University of Porto and Instituto Nacional Of Saude Dr Ricardo Jorge Insa
Type: Journal Article | Journal: Journal of applied toxicology : JAT | Year: 2015

Abuse of synthetic drugs is widespread worldwide. Studies indicate that piperazine designer drugs act as substrates at dopaminergic and serotonergic receptors and/or transporters in the brain. This work aimed to investigate the cytotoxicity of N-benzylpiperazine, 1-(3-trifluoromethylphenyl)piperazine, 1-(4-methoxyphenyl)piperazine and 1-(3,4-methylenedioxybenzyl)piperazine in the differentiated human neuroblastoma SH-SY5Y cell line. Cytotoxicity was evaluated after 24h incubations through the MTT reduction and neutral red uptake assays. Oxidative stress (reactive oxygen and nitrogen species production and glutathione content) and energetic (ATP content) parameters, as well as intracellular Ca(2+), mitochondrial membrane potential, DNA damage (comet assay) and cell death mode were also evaluated. Complete cytotoxicity curves were obtained after 24h incubations with each drug. A significant decrease in intracellular total glutathione content was noted for all the tested drugs. All drugs caused a significant increase of intracellular free Ca(2+) levels, accompanied by mitochondrial hyperpolarization. However, ATP levels remained unchanged. The investigation of cell death mode revealed a predominance of early apoptotic cells. No genotoxicity was found in the comet assay. Among the tested drugs, 1-(3-trifluoromethylphenyl)piperazine was the most cytotoxic. Overall, piperazine designer drugs are potentially neurotoxic, supporting concerns on risks associated with the abuse of these drugs.

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